In vitro effects of heat extracts from firefly squid, Watasenia scintillans, (FSE-1) and desalted FSE-1 (DFSE-1) on the activities of phagocytes and lymphocyte proliferation of rainbow trout were evaluated. In the first experiment, the effects of FSE-1 and DFSE-1 were compared by measuring respiratory burst and potential killing activities of blood phagocytes, and by measuring lymphoblastic transformation level with or without Con A. In this experiment, DFSE-1 was found more effective than FSE-1. In the second experiment, effects of different doses of DFSE-1 were examined by measuring nitro blue tetrazorium (NBT) reduction level, respiratory burst activity and potential killing activity of head kidney macrophages and lymphocyte proliferation test with Con A or LPS. When diluted (×1-×8) DFSE-1 was added, the head kidney macrophages showed significant (p<0.05) elevation of NBT reduction, respiratory burst and potential killing activities compared to the control. The lymphoblastic transformation rate was significantly (p<0.05) higher than that of control when diluted (×1-×16) DFSE-1 was added with Con A or LPS. These results suggest that DFSE-1 administration effectively stimulate the defense mechanism of rainbow trout.
A metalloprotease was purified from a culture supernatant of the fish pathogen, Listonella (Vibrio) anguillarum. The molecular weight of the enzyme was estimated to be 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was inhibited by EDTA and o-phenanthroline.Zn2+ and Mn2+ at 10μM reactivated the enzyme which had been inactivated with 0.1μM EDTA.Ca2+ at 1mM enhanced the enzyme activity by 170% and increased stability of the enzyme activity at high temperatures. One noteworthy finding is that the enzyme hydrolyzed only butyloxycarbonyl-leucyl-seryl-threonyl-arginyl-4-methylcoumaryl-7-amide (Boc-Leu-Ser-Thr-Arg-MCA, a substrate for plasma activated protein C (APC), an anticoagulation factor) among the 17 peptydyl-4-methylcoumaryl-7-amide. The enzyme extended the clotting time of rainbow trout plasma. The enzyme enhanced the hydrolyzing activity of Boc-Leu-Ser-Thr-Arg-MCA in trout plasma. These results indicate that the enzyme possesses an activity similar to APC and activates protein C of rainbow trout. The enzyme inhibited activities of Factor Xa and thrombin. From these findings, it was concluded that the metalloprotease produced by L. anguillarum plays a role as an anticoagulation factor similar to APC.
Seasonal occurrence of Myxobolus artus infection in common carp Cyprinus carpio was monitored in a fry pond (Tokyo Metropolis) for one and a half year. Formation of pseudocysts in the skeletal muscle was detected from August, and the prevalence of infection reached a maximum of 10% in September. After maturation of pseudocysts, diseased fish released spores into the water at a maximum level of about 3×106 spores/ day/fish in October. Spore release decreased in late autumn and winter, increased in spring and exhausted in the following summer in 1-year old recovered fish. Mortality of diseased fish was negligible in September. However, chronic mortality with a typical sign of anemic gills was found during the spore releasing period. During a high level period of spore discharge, hematological indices (Ht, Hb, and RBC) in diseased fish significantly decreased, and the percentage of immature erythrocytes increased. Histopathological examination showed that numerous phagocytosed spores caused local occlusion and destruction of gill capillaries and exfoliation of gill epithelium, suggesting blood loss from the damaged gills. It was suggested that chronic hemorrhagic anemia was associated with spore discharge from carp heavily infected with M. artus.
On the basis of the 16S rRNA sequence data analysis among the closely related species, the specific primers for Flexibacter maritimus, Flavobacterium branchiophilum, and Cytophaga columnaris were constructed. The specificity in amplifying the 16S rDNA of each species was confirmed by using selected strains of related bacteria and principal fish pathogenic bacteria. A primer pair of MAR 1 and MAR2 could differentiate F. maritimus from other species. The PCR performed with a pair of primers specific to F. branchiophilum failed to amplify the 16S rDNA. However, the PCR with a pair of a specific primer BRA1 and a universal primer 1500R succeeded in specifically amplifying 16S rDNA from F. branchiophilum. The Nucleotide Sequence Database (GenBank) contains two different 16S rRNA sequence data for C. columnaris. On the basis of these data, we produced two primer pairs. A pair of COL1 and 1500R amplified the 16S rDNA from 5 of 7 strains of C. columnaris, and a pair of COLa and COLb worked for the rest of the strains. In addition, these two groups within C. columnaris were supported by PCR-RFLP.
Since 1991, mortalities have occurred in 0-year-old Japanese flounder Paralichthys olivaceus (2-22g body weight) cultured in Oita and Ehime prefectures from September to December when the water temperature ranged from 17 to 24°C. The diseased fish showed dark coloration and swam inactively, but no definite symptom of the disease was observed. Daily and the cumulative mortalities were 0.01-0.6% and 0.6-4.8%, respectively. A bacterium was purely isolated from the kidney of diseased fish and identified as Pasteurella piscicida by the biological, biochemical and serological characteristics. The isolates of the bacterium were highly sensitive to most of antibacterial agents tested (oxytetracycline, ampicillin, oxolinic acid, flumequine, florfenicol, bicozamycin and fosfomycin). Oral administration of oxytetracycline hydrochloride was effective to control the epizootics. The experimental infection by intraperitoneal injection at about 18°C revealed that the bacterium was pathogenic for juvenile Japanese flounder.
Morphology and property of viral nucleic acid of RV-PJ (rod-shaped nuclear virus of Penaeus japonicus), which causes mass mortality of kuruma shrimp (P.japonicus) were described. The virions of causative virus were long ovoid-shaped, and contained a partially lenticular-shaped cylindrical nucleocapsid measuring 84 nm × 226 nm within the loosely surrounding envelope. Nucleic acid from the purified virus particles were detected as a single band on a 0.8% agarose gel which was resistant to RNaseA and was digested with restriction endonucleases such as EcoRI, indicating that the viral genome consists of a non-segmented, double-stranded DNA molecule of approximately 163 kbp. These characteristics of the virus suggest that RV-PJ should be classified in the same group with unassigned rod-shaped ds-DNA viruses of invertebrates such as Oryctes rhinoceros virus, which were previously classified in the Nudibaculovirinae, a subfamily of the Baculoviridae. Therefore, we re-designate the virus as Penaeid rod-shaped DNA virus (PRDV). Furthermore, we propose to name the disease which caused by this virus as penaeid acute viremia (PAV).
Mass mortality occurred in dusky spinefoot, Siganus fuscescens, captured at Urasoko Bay and reared in a tank at Fukui Prefectural Fisheries Experimental Station. Diseased fish showed dark body color and hemorrhage in the mouth and eyes and at the base of the pectoral and ventral fins. The bacterium isolated from the diseased fish was found to be pathogenic to dusky spinefoot by intraperitoneal injection. From morphological and biochemical characteristics, the isolate was identified as Streptococcus iniae.